1. Field of the Invention
The present invention relates to a method of synthesis of .beta.-lactam antibiotics.
2. Discussion of the Background
It has long been known that beta-lactam antibiotics can be formed from their respective nucleus and side-chain components via enzymatic pathways (C. A. Claridge et al., Nature, Vol. 187;237, 1960).
From more recent studies it is known that a beta-lactam antibiotic, for example amoxicillin, synthesized via enzymatic pathways, has higher purity and accordingly lower toxicity than amoxicillin synthesized by chemical pathways (PCT WO 94/17800).
The general interest for industrially practical processes for enzymatically synthesizing beta-lactam antibiotics is correspondingly great.
In order to achieve this objective, a series of technical problems must be overcome or needs fulfilled, especially:
a) The need to maximize the desired synthesis reaction catalyzed by the enzyme and to minimize the undesired side reactions catalyzed by the same enzyme, such as hydrolysis of the activated side-chain components and of the beta-lactam antibiotic (see the reaction mechanism below). PA1 b) The need to isolate the product in a way which permits reuse of the biocatalyst, or, in other words, the enzyme. PA1 1. The activity measured for the first use of a covalently immobilized enzyme is usually much too high, because free enzyme is often still present. This free enzyme is adsorbed non-covalently on the support material, thus giving a false impression of higher activity of the "immobilized" enzyme, specifically until it has been eliminated by interaction with the substrate and by washing processes, such elimination lasting at least one cycle of use and usually several cycles. PA1 2. Substrate and product molecules can be adsorbed on the support material on which the enzyme is covalently immobilized, and thus can elude analytical determination thereby falsifying the balance between synthesis and hydrolysis reactions. PA1 3. The immobilized enzyme almost always loses activity in the course of its use, but especially during the first three cycles of use.
Reaction mechanism ##STR1##
Even now are numerous patent applications which have already been published, as well as patents granted, which claim decisive advances in solving these problems and which are based on experimental findings obtained with enzymes immobilized on insoluble carrier particles, usually covalently; in other words with systems, developed in the most recent decades, for reuse of enzymes.
It is, therefore, self-evident that the operational stability (O-ST) of these immobilized enzymes must be studied under practical conditions, in order to obtain an appraisal of the industrial productivity of such a biocatalyst.
In most cases, however, investigators have been satisfied with a minimum of tests, namely with testing of a single batch.
The result obtained in this way was then often generalized in a global claim, without regard to a fact well-known to the experts, that a result obtained from only a single batch is usually misleading for characterization of an immobilized enzyme and a reaction catalyzed thereby, specifically for the following reasons:
In this connection, it must be recalled that immobilized enzymes are employed mainly because of their ready reusability, or in other words, their usability over many batch cycles, and because of the purity of the products which have been synthesized under these conditions.
The purity laws automatically preclude contamination by free enzyme; therefore, the product from the first batch should not be used in any case for the synthesis of pharmaceuticals.
This phenomenon is relevant in particular for the first batch. Thereafter a kind of stable state is established by saturation of the adsorbing support surface. In this context, analytical methods which consider only the dissolved constituents are misleading. In order to achieve a correct balance, only methods which permit solubilization of all molecules present should be used.
For all of these reasons it is advisable to run at least four successive batch cycles in order to obtain a somewhat correct estimate of product yield, degree of conversion of the reaction and O-ST of the biocatalyst.
The test methods must obviously be significant, meaning that the product must be isolated if possible and its composition must be studied by, for example, HPLC or NM R. Samples taken from the heterogeneous product or reaction mixture must be solubilized before they are tested.
These rules have not been followed, however, in most of the patent applications and granted patents. To the knowledge of the present inventors, there is currently only a single application (PCT WO 96/23897, Chemferm, Boesten ct al.) which provides data on the O-ST of the biocatalyst obtained by testing of five successive batch cycles, with a degree of conversion of about 40% of the nucleus components.
Another critical aspect in the field of enzymatic synthesis of beta-lactam antibiotics relates to isolation of the product. Isolation of the product is problematic for the following reasons:
The antibiotic synthesized in this way is normally obtained as a heterogeneous mixture of solid and dissolved product, which must be separated from the biocatalyst, or in other words, the immobilized enzyme, which is also solid. In this regard, Clausen et al. (PCT WO 96/02663 A1) have published a process for synthesis of beta-lactam antibiotics at constantly high concentration of the reactants, wherein the desired product is obtained mainly as a solid product in the form of small crystals, which are continuously separated from the reactor by passing them through a sieve, which holds up the biocatalyst, into a centrifuge, in which such crystals are isolated. The centrifuge supernatant is pumped back into the reactor via a tank, in which further substrate is added. This process offers a noteworthy advance, in that the product is removed rapidly from the reaction medium via crystallization and centrifugation, and consequently cannot be hydrolyzed to undesired secondary products.
Despite this advantage, the process does not permit problem-free continuous operation in the stirred reactor.
The risk exists that in this process, which in principle is a wet-sieve process, the bottom sieve of the reactor will gradually become fouled because of the non-uniformity of grain sizes, and that the accumulation of solid products will form in the reactor itself a pasty mass at first and ultimately a solid conglomerate of product and catalyst particles, thus making further operation of the reactor impossible.
In summary, it can be said that the prior art known prior to this invention leaves unsolved the true problem, namely the clean separation of the product in combination with reusability, meaning reusability of the biocatalyst. A need, therefore, continues to exist for such a process which can be achieved without problems in the stirred reactor or-in the fixed-bed reactor.